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The goal of this work was to develop and validate an analytical method for the detection and quantification of thebiogenicaminesserotonin(5-HT),dopamine(DA)andnorepinephrine(NE),usingmicroextractioninpacked syringe (MEPS) andliquid chromatography coupled to electrochemical detection (HPLC-ED) in urine. The method was validated according to inter-nationally accepted guidelines from the Food and Drug Administration. Linearity was established between 50 and 1000 ng/mLfor5-HTand between5and 1000ng/mLforDAand NE, withdeterminationcoefficients(R 2 )>0.99forall compounds.Thelimitsof quantification and detection were respectively 50 and 20 ng/mL for 5-HT, and 5 and 2 ng/mL for DA and NE. Within- andbetween-runprecision ranged from0.84 to 9.41%, while accuracy ranged from0.79 to 12.76%for all compounds. The interme-diate precision and accuracy were 1.50–8.36 and 0.54–13.51%, respectively. The method was found suitable for clinicalroutine analysis of the studied compounds, using a sample volume of 0.5 mL. This is thefirst study employing a commer-cially available MEPS column for the simultaneous detection and quantification of 5-HT, DA and NE in urine by coulometricdetection. Copyright © 2012 John Wiley & Sons, Ltd.Keywords: biogenic amines; urine; MEPS; HPLC-ED

The main scope of this work is the evaluation and potential application of hydrophobic interaction chromatography in the isolation of recombinant human soluble catechol-O-methyltransferase (hSCOMT) from an Escherichia coli cell extract. Therefore, a comparative study on the interaction of recombinant hSCOMT with different hydrophobic adsorbents (butyl-, octyl-, phenyl- and epoxy-Sepharose), was developed. The four adsorbents were evaluated in terms of selectivity, recovery and fractionation of recombinant hSCOMT from its Escherichia coli-free culture broth. Our data shows that the adjustment of the ionic strength on the mobile phase and the type of hydrophobic ligand are the most useful factors for a complete binding of hSCOMT and a selective fractionation of contaminating proteins. The results of these studies demonstrate that, although epoxy-Sepharose is used as a last resort due to the high salt concentrations needed, hSCOMT bind to the other three resins at low concentrations of ammonium sulfate (≤0.6 m) and eluted just by decreasing the ionic strength on the eluent to 0 m, without loss of specific of activity. The stepwise gradient with 0.6, 0.2, 0.075 and 0 m of ammonium sulfate onto a butyl-Sepharose media was found to be the most effective in the isolation of hSCOMT, leading to an enzyme solution with a 3.9-fold increased in specific activity. Since biochemical and structural studies require significant quantities of the enzyme in an active form, the above described approach may give some insight into the optimization and development of new purification strategies of mammalian COMTs. Copyright © 2007 John Wiley & Sons, Ltd.

The enzyme catechol-O-methyltransferase (COMT) plays an important role in the metabolism of catechol estrogens and degradation of the catecholamine neurotransmitters, such as epinephrine. Several analytical methods, mainly high-performance liquid chromatography with electrochemical amperometric detection, have been reported for the analysis of catecholamines and their metabolites in biological fluids. In this paper we report the relevance of controlling temperature in calibration procedures of metanephrine, an O-methylated product of catechol-O-methyltransferase, using epinephrine as substrate. The results at higher temperatures show shorter retention times of metanephrine, no undue band-broadening and increased electro signals. This study also showed that, despite different temperatures leading to similarly specific activities of recombinant human COMT as expected, there are additional advantages in flow analytical methods where good sensitivity, efficiency and selectivity is required, mainly in tissues with low levels of COMT activity.